LOCUS       sequence-203712-        7130 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression plasmids for production of recombinant 
            metallothionein 2a K30/31A mutant.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7130)
  AUTHORS   Singh AK, Pomorski A, Wu S, Peris-Diaz MD, Czepczynska-Krezel H, 
            Krezel A
  TITLE     The connection of alpha- and beta-domains in mammalian 
            metallothionein-2 differentiates Zn(II) binding affinities, affects 
            folding, and determines zinc buffering properties.
  JOURNAL   Metallomics. 2023 Jun 1;15(6):mfad029. doi: 10.1093/mtomcs/mfad029.
  PUBMED    37147085
REFERENCE   2  (bases 1 to 7130)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: 
            "Metallomics."; date: "2023-06-1"; volume: "15(6):mfad029. doi"; 
            pages: " 10.1093/mtomcs/mfad029"
COMMENT     Sequence Label: MT2a_K30A K31A
FEATURES             Location/Qualifiers
     source          1..7130
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        15..119
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(120..188,189..980)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(338..357)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      complement(1022..1535)
                     /direction=LEFT
                     /label=M13 ori
                     /note="M13 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1217..1238)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     1429..1448
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     rep_origin      1646..2234
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2135..2154
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2388..2405
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     complement(2427..2446)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2587..2609
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2603..2794)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(3351..4433)
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=lacI
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     4395..4414
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4434..4511)
                     /gene="lacI"
                     /label=lacI promoter
     terminator      4664..4750
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      4847..4933
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     5068..5087
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5068..5086
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5087..5111
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     RBS             5126..5148
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             5201..6019
                     /codon_start=1
                     /gene="Saccharomyces cerevisiae VMA1"
                     /product="5' region of the intein from the yeast Vma1 
                     subunit of the vacuolar ATPase (Chong et al., 1998)"
                     /label=Sce VMA intein 5' region
                     /translation="CFAKGTNVLMADGSIECIENIEVGNKVMGKDGRPREVIKLPRGRE
                     TMYSVVQKSQHRAHKSDSSREVPELLKFTCNATHELVVRTPRSVRRLSRTIKGVEYFEV
                     ITFEMGQKKAPDGRIVELVKEVSKSYPISEGPERANELVESYRKASNKAYFEWTIEARD
                     LSLLGSHVRKATYQTYAPILYENDHFFDYMQKSKFHLTIEGPKVLAYLLGLWIGDGLSD
                     RATFSVDSRDTSLMERVTEYAEKLNLCAEYKDRKEPQVAKTVNLYSKVVRG"
     CDS             6026..6178
                     /codon_start=1
                     /gene="Bacillus circulans chiA"
                     /product="chitin binding domain from chitinase A1 (Watanabe
                     et al., 1994)"
                     /label=CBD
                     /translation="TNPGVSAWQVNTAYTAGQLVTYNGKTYKCLQPHTSLAGWEPSNVP
                     ALWQLQ"
     CDS             6182..6730
                     /codon_start=1
                     /gene="Saccharomyces cerevisiae VMA1"
                     /product="modified 3' region of the intein from the yeast 
                     Vma1 subunit of the vacuolar ATPase (Chong et al., 1998)"
                     /label=Sce VMA intein 3' region
                     /note="C-terminal cleavage of this intein can be induced by
                     thiols."
                     /translation="GHGGIRNNLNTENPLWDAIVGLGFLKDGVKNIPSFLSTDNIGTRE
                     TFLAGLIDSDGYVTDEHGIKATIKTIHTSVRDGLVSLARSLGLVVSVNAEPAKVDMNVT
                     KHKISYAIYMSGGDVLLNVLSKCAGSKKFRPAPAAAFARECRGFYFELQELKEDDYYGI
                     TLSDDSDHQFLLGSQVVVQN"
     primer_bind     complement(7024..7042)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      7038..7085
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
ORIGIN
        1 ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt
       61 atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagta
      121 tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg
      181 tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac
      241 gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccg
      301 aagaacgttt cccaatgatg agcactttta aagttctgct atgtggcgcg gtattatccc
      361 gtgttgacgc cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg
      421 ttgagtactc accagtcaca gaaaagcatc ttacggatgg catgacagta agagaattat
      481 gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg acaacgatcg
      541 gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg
      601 atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc
      661 ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg cgaactactt actctagctt
      721 cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca cttctgcgct
      781 cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag cgtgggtctc
      841 gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgtatcgta gttatctaca
      901 cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct
      961 cactgattaa gcattggtaa ctgtcagacc aagtttactc atatatactt tagattgatt
     1021 taccccggtt gataatcaga aaagccccaa aaacaggaag attgtataag caaatattta
     1081 aattgtaaac gttaatattt tgttaaaatt cgcgttaaat ttttgttaaa tcagctcatt
     1141 ttttaaccaa taggccgaaa tcggcaaaat cccttataaa tcaaaagaat agcccgagat
     1201 agggttgagt gttgttccag tttggaacaa gagtccacta ttaaagaacg tggactccaa
     1261 cgtcaaaggg cgaaaaaccg tctatcaggg cgatggccca ctacgtgaac catcacccaa
     1321 atcaagtttt ttggggtcga ggtgccgtaa agcactaaat cggaacccta aagggagccc
     1381 ccgatttaga gcttgacggg gaaagccggc gaacgtggcg agaaaggaag ggaagaaagc
     1441 gaaaggagcg ggcgctaggg cgctggcaag tgtagcggtc acgctgcgcg taaccaccac
     1501 acccgccgcg cttaatgcgc cgctacaggg cgcgtaaaag gatctaggtg aagatccttt
     1561 ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc
     1621 ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct
     1681 tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa
     1741 ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact gtccttctag
     1801 tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc
     1861 tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg
     1921 actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca
     1981 cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat
     2041 gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg
     2101 tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc
     2161 ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc
     2221 ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc
     2281 cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg
     2341 cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga
     2401 gcgaggaagc tatggtgcac tctcagtaca atctgctctg atgccgcata gttaagccag
     2461 tatacactcc gctatcgcta cgtgactggg tcatggctgc gccccgacac ccgccaacac
     2521 ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc cgcttacaga caagctgtga
     2581 ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgaggc
     2641 agctgcggta aagctcatca gcgtggtcgt gcagcgattc acagatgtct gcctgttcat
     2701 ccgcgtccag ctcgttgagt ttctccagaa gcgttaatgt ctggcttctg ataaagcggg
     2761 ccatgttaag ggcggttttt tcctgtttgg tcactgatgc ctccgtgtaa gggggatttc
     2821 tgttcatggg ggtaatgata ccgatgaaac gagagaggat gctcacgata cgggttactg
     2881 atgatgaaca tgcccggtta ctggaacgtt gtgagggtaa acaactggcg gtatggatgc
     2941 ggcgggacca gagaaaaatc actcagggtc aatgccagcc gaacgccagc aagacgtagc
     3001 ccagcgcgtc ggccgccatg ccggcgataa tggcctgctt ctcgccgaaa cgtttggtgg
     3061 cgggaccagt gacgaaggct tgagcgaggg cgtgcaagat tccgaatacc gcaagcgaca
     3121 ggccgatcat cgtcgcgctc cagcgaaagc ggtcctcgcc gaaaatgacc cagagcgctg
     3181 ccggcacctg tcctacgagt tgcatgataa agaagacagt cataagtgcg gcgacgatag
     3241 tcatgccccg cgcccaccgg aaggagctga ctgggttgaa ggctctcaag ggcatcggtc
     3301 gagatcccgg tgcctaatga gtgagctaac ttacattaat tgcgttgcgc tcactgcccg
     3361 ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga
     3421 gaggcggttt gcgtattggg cgccagggtg gtttttcttt tcaccagtga gacgggcaac
     3481 agctgattgc ccttcaccgc ctggccctga gagagttgca gcaagcggtc cacgctggtt
     3541 tgccccagca ggcgaaaatc ctgtttgatg gtggttaacg gcgggatata acatgagctg
     3601 tcttcggtat cgtcgtatcc cactaccgag atgtccgcac caacgcgcag cccggactcg
     3661 gtaatggcgc gcattgcgcc cagcgccatc tgatcgttgg caaccagcat cgcagtggga
     3721 acgatgccct cattcagcat ttgcatggtt tgttgaaaac cggacatggc actccagtcg
     3781 ccttcccgtt ccgctatcgg ctgaatttga ttgcgagtga gatatttatg ccagccagcc
     3841 agacgcagac gcgccgagac agaacttaat gggcccgcta acagcgcgat ttgctggtga
     3901 cccaatgcga ccagatgctc cacgcccagt cgcgtaccgt cttcatggga gaaaataata
     3961 ctgttgatgg gtgtctggtc agagacatca agaaataacg ccggaacatt agtgcaggca
     4021 gcttccacag caatggcatc ctggtcatcc agcggatagt taatgatcag cccactgacg
     4081 cgttgcgcga gaagattgtg caccgccgct ttacaggctt cgacgccgct tcgttctacc
     4141 atcgacacca ccacgctggc acccagttga tcggcgcgag atttaatcgc cgcgacaatt
     4201 tgcgacggcg cgtgcagggc cagactggag gtggcaacgc caatcagcaa cgactgtttg
     4261 cccgccagtt gttgtgccac gcggttggga atgtaattca gctccgccat cgccgcttcc
     4321 actttttccc gcgttttcgc agaaacgtgg ctggcctggt tcaccacgcg ggaaacggtc
     4381 tgataagaga caccggcata ctctgcgaca tcgtataacg ttactggttt cacattcacc
     4441 accctgaatt gactctcttc cgggcgctat catgccatac cgcgaaaggt tttgcgccat
     4501 tcgatggtgt ccgggatctc gacgctctcc cttatgcgac tcctgcatta ggaagcagcc
     4561 cagtagtagg ttgaggccgt tgagcaccgc cgccgcaagg aatggtgcat gccggcatgc
     4621 cgccctttcg tcttcaagaa ttaattccca attccccagg catcaaataa aacgaaaggc
     4681 tcagtcgaaa gactgggcct ttcgttttat ctgttgtttg tcggtgaacg ctctcctgag
     4741 taggacaaat ccgccgggag cggatttgaa cgttgcgaag caacggcccg gagggtggcg
     4801 ggcaggacgc ccgccataaa ctgccaggaa ttaattcccc aggcatcaaa taaaacgaaa
     4861 ggctcagtcg aaagactggg cctttcgttt tatctgttgt ttgtcggtga acgctctcct
     4921 gagtaggaca aatccgccgg gagcggattt gaacgttgcg aagcaacggc ccggagggtg
     4981 gcgggcagga cgcccgccat aaactgccag gaattgggga tcggaattaa ttcccggttt
     5041 aaaccgggga tctcgatccc gcgaaattaa tacgactcac tataggggaa ttgtgagcgg
     5101 ataacaattc ccctctagaa ataattttgt ttaactttaa gaaggagata tacttatgaa
     5161 aatcgaagaa ggtaaactgg taatcggttc cctggagggt tgctttgcca agggtaccaa
     5221 tgttttaatg gcggatgggt ctattgaatg tattgaaaac attgaggttg gtaataaggt
     5281 catgggtaaa gatggcagac ctcgtgaggt aattaaattg cccagaggaa gagaaactat
     5341 gtacagcgtc gtgcagaaaa gtcagcacag agcccacaaa agtgactcaa gtcgtgaagt
     5401 gccagaatta ctcaagttta cgtgtaatgc gacccatgag ttggttgtta gaacacctcg
     5461 tagtgtccgc cgtttgtctc gtaccattaa gggtgtcgaa tattttgaag ttattacttt
     5521 tgagatgggc caaaagaaag cccccgacgg tagaattgtt gagcttgtca aggaagtttc
     5581 aaagagctac ccaatatctg aggggcctga gagagccaac gaattagtag aatcctatag
     5641 aaaggcttca aataaagctt attttgagtg gactattgag gccagagatc tttctctgtt
     5701 gggttcccat gttcgtaaag ctacctacca gacttacgct ccaattcttt atgagaatga
     5761 ccactttttc gactacatgc aaaaaagtaa gtttcatctc accattgaag gtccaaaagt
     5821 acttgcttat ttacttggtt tatggattgg tgatggattg tctgacaggg caactttttc
     5881 ggttgattcc agagatactt ctttgatgga acgtgttact gaatatgctg aaaagttgaa
     5941 tttgtgcgcc gagtataagg acagaaaaga accacaagtt gccaaaactg ttaatttgta
     6001 ctctaaagtt gtcagaggtg ctagcacaaa tcctggtgta tccgcttggc aggtcaacac
     6061 agcttatact gcgggacaat tggtcacata taacggcaag acgtataaat gtttgcagcc
     6121 ccacacctcc ttggcaggat gggaaccatc caacgttcct gccttgtggc agcttcaagg
     6181 tggccacggt ggtattcgca ataatcttaa tactgagaat ccattatggg acgctattgt
     6241 tggcttagga ttcttgaagg acggtgtcaa aaatattcct tctttcttgt ctacggacaa
     6301 tatcggtact cgtgaaacat ttcttgctgg tctaattgat tctgatggct atgttactga
     6361 tgagcatggt attaaagcaa caataaagac aattcatact tctgtcagag atggtttggt
     6421 ttcccttgct cgttctttag gcttagtagt ctcggttaac gcagaacctg ctaaggttga
     6481 catgaatgtc accaaacata aaattagtta tgctatttat atgtctggtg gagatgtttt
     6541 gcttaacgtt ctttcgaagt gtgccggctc taaaaaattc aggcctgctc ccgccgctgc
     6601 ttttgcacgt gagtgccgcg gattttattt cgagttacaa gaattgaagg aagacgatta
     6661 ttatgggatt actttatctg atgattctga tcatcagttt ttgcttggat ctcaggttgt
     6721 tgtacagaac atggatccga actgcagctg tgcggcgggt gattcttgca cgtgtgcggg
     6781 cagctgcaaa tgcaaagaat gcaaatgcac cagctgcgca gcaagctgct gcagctgctg
     6841 tccggtgggc tgtgcgaaat gcgcgcaagg ctgcatttgc aaaggcgcga gcgataaatg
     6901 cagctgctgc gcgtaactgc agaccaccat cgtcgacgga tccgaattcc ctgcaggtaa
     6961 ttaaataact agttgatccg gctgctaaca aagcccgaaa ggaagctgag ttggctgctg
     7021 ccaccgctga gcaataacta gcataacccc ttggggcctc taaacgggtc ttgaggggtt
     7081 ttttgctgaa aggaggaact atatccggat aactacgtca ggtggcactt
//
